RING fingers mediate ubiquitin-conjugating enzyme (E2)-dependent ubiquitination

Abstract

A RING finger-containing protein (AO7) that binds ubiquitin-conjugating enzymes (E2s) and is a substrate for E2-dependent ubiquitination was identified. Mutations of cation-coordinating residues within AO7's RING finger abolished ubiquitination, as did chelation of zinc. Several otherwise-unrelated RING finger proteins, including BRCA1, Siah-1, TRC8, NF-X1, kf-1, and Praja1, were assessed for their ability to facilitate E2-dependent ubiquitination. In all cases, ubiquitination was observed. The RING fingers were implicated directly in this activity through mutations of metal-coordinating residues or chelation of zinc. These findings suggest that a large number of RING finger-containing proteins, with otherwise diverse structures and functions, may play previously unappreciated roles in modulating protein levels via ubiquitination.

Figure 1

AO7 is involved in E2-dependent ubiquitination in vitro. (A) Equimolar amounts of the indicated GS-bound fusion proteins or GST alone were incubated in the presence of E1, E2 (UbcH5B), proteins from bacterial lysates, and ³²P-labeled Ub for 1.5 h at 30°C. The entire reaction was resolved under reducing conditions by SDS/PAGE. (B) GST fusion proteins were incubated as described for A in the absence or presence of E2 (UbcH5B). GST fusions of AO7T, Nedd4, and E6-AP migrate at approximately 69, 120, and 115 kDa, respectively.

Figure 2

A region required for E2-dependent ubiquitination. (A) Schematic representation of AO7 and AO7 truncations fused to GST at their N termini. The RING and positions of cysteines (C) are indicated. (B) Equimolar amounts of the indicated GST fusions were evaluated for ubiquitination by using ³²P-labeled Ub (Upper) or for binding to ³⁵S-labeled UbcH5B (Lower). Upper and Lower are from separate experiments.

Figure 3

Ubiquitination of AO7 depends on RING-H2 and zinc. (A) Schematic of AO7 and RING-H2 domain. Cysteines are numbered. Eight residues predicted to be crucial to Zn²⁺ coordination are indicated by lines connecting AO7 and the RING consensus (16). Variable loops between the second and third (Loop 1) and between the sixth and seventh (Loop 2) predicted Zn²⁺ coordinating residues are indicated. (B and C) GST fusion proteins were assayed for ubiquitination (Upper) and binding of labeled UbcH5B (Lower). Numbers above lanes denote residues that were mutated. (C, lanes 6–8) Labeling indicates amino acids to which H157 and C158 were changed. Upper and Lower are from separate experiments. (D) COS-7 cells were transfected with plasmid encoding HA-tagged AO7 (HA-AO7), a RING mutant (HA-AO7RM), or empty vector (pcDNA3). HA-tagged proteins were immunoprecipitated and resolved by SDS/12% PAGE. The membrane was probed with anti-HA (Upper) or polyclonal anti-UbcH5B (Lower). (E) Anti-HA immunoprecipitates of cells transfected as described for D were resolved by SDS/8% PAGE. The membrane was probed with anti-Ub. (F) GS-bound GST-AO7T and GST-Nedd4 were incubated with the indicated chelators before assaying ubiquitination. Data are normalized to samples not exposed to chelators. (G) TPEN chelation of GS-bound GST-AO7T was followed by incubation with ZnCl₂ for 3 h. GS was washed extensively before evaluation of ubiquitination.

Figure 4

Evaluation of four RING-H2 proteins. (A) Alignment of RING-H2 regions of proteins. Loops and predicted coordination sites are indicated above. kf-1 has two possible alignments beginning with either C618 or C621; the alignment shown begins with C621. Full-length forms of proteins are schematized below. Heavy lines indicate regions fused to GST (Praja1 is full length), and RING finger regions are indicated (▧). TRC8 is a multispanning membrane protein. (B) GST-Praja1 and mutants of the fifth putative coordination site to either Ser (H → S) or Cys (H → C) were evaluated for ubiquitination. (C) GST fusions of the indicated proteins were evaluated as described for B. The kf-1, NF-X1, and TRC8 fusions included amino acids 538–686, 207–645, and 477–664, respectively. The C → S mutation of kf-1 was at amino acid 621; NF-X1 C → S was a double mutation at C342 and C345. Because of difficulty expressing GST-NF-X1, only 5 pmol was used, and 20 pmol of NF-X1 C → S was used. (D) Soluble material was removed from ubiquitination reactions carried out as described for C, and GS beads were washed extensively. Both fractions were resolved by SDS/PAGE. (E) ³⁵S-labeled Praja1 was resolved directly on SDS/PAGE (Input) or subjected to a ubiquitination reaction in E1-containing reaction buffer with or without UbcH5B. (F) GS-bound proteins were evaluated for ubiquitination with either wild-type Ub or UbK₀. After Western transfer, membranes were immunoblotted with anti-Ub. GST-Praja1 and GST-kf-1 migrate at approximately 85 kDa and 50 kDa, respectively.

Figure 5

RING-HC proteins are also involved in ubiquitination. (A) A GST fusion of amino acids 1–788 of mouse BRCA1 was evaluated for ubiquitination. (B) GS-bound GST-BRCA1 was incubated with (+) or without (−) TPEN, followed by washing of GS before a ubiquitination reaction. (C) Flag-tagged Siah-1 (Siah-1) or a mutant in the third coordination site (Siah-1 C → S) was translated with [³⁵S]Met in wheat germ lysate. Material was resolved directly on SDS/PAGE (Input) or evaluated for ubiquitination with or without UbcH5B. (D) GST fusions of Siah-1, the C → S Siah-1 mutant, or RING-deleted (Siah-1ΔR; ref. 12) were evaluated for ubiquitination.